In addition to the major endogenous estrogens, specific estrogen metabolites (including 16alpha(OH) estrone, 4-(OH) estrone and 4-(OH) estradiol) are thought to be genotoxic and potentially carcinogenic in humans. Despite the potential importance of endogenous estrogen metabolites in breast carcinogenesis, few studies have investigated their association with human breast cancer risk. The few epidemiological studies of estrogen metabolism and breast cancer have reported only 16alpha(OH) estrone and 2-(OH) estrone excretion or the ration between the two. The reasons for this lack of data are the difficulty and expense of estrogen metabolite analysis. Development of an inexpensive, rapid method will enable the performance of large epidemiological studies to determine the associations between specific estrogen metabolites and breast cancer risk, as well as the associations between estrogen metabolites and modifiable risk factors such as obesity and diet. The main purpose of this project is to develop a rapid, inexpensive method of analysis of estrogen metabolites, that will be practical for use in large epidemiological studies. At this time, the GC-MS "gold standard" method is very labor intensive and much too expensive for use in such studies. An HPLC-MS/MS method should provide similar quality of data at a much lower cost. The specific aims of this project are to develop an HPLC-MS/MS method for analysis of urinary estrogens and estrogen metabolites, and to validate this method against the "gold standard" GC-MS method. Urine from the mid-follicular and mid-luteal phases of the menstrual cycle, a well as from post-menopausal women, will be analyzed for both methods for determination of quality control parameters. The development of this method will enable the performance of large epidemiological studies of estrogen metabolism and breast cancer.